Bone is subject to constant breakdown and re-synthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned.
There is a plethora of conditions which are characterized by the need to enhance bone formation or to inhibit bone resorption. Perhaps the most obvious is the case of bone fractures, where it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in facial reconstruction procedures. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.
Bone fractures are still treated exclusively using casts, braces, anchoring devices and other strictly mechanical means. Further bone deterioration associated with post-menopausal osteoporosis has been treated with estrogens or bisphosphonates, which may have drawbacks for some individuals. Treatment of bone or other skeletal disorders, such as those associated with cartilage, can be achieved either by enhancing bone formation or inhibiting bone resorption or both.
Bone tissue is an excellent source for factors which have the capacity for stimulating bone cells. Thus, extracts of bovine bone tissue obtained from slaughterhouses contain not only structural proteins which are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors which can stimulate bone cells to proliferate. Among these latter factors are transforming growth factor β, the heparin-binding growth factors (e.g., acidic and basic fibroblast growth factor), the insulin-like growth factors (e.g., insulin-like growth factor I and insulin-like growth factor II), and a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other types of cells, as well as on bone cells.
The cells which are responsible for forming bone are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase. They also synthesize a number of growth regulatory peptides which are stored in the bone matrix, and are presumably responsible for normal bone formation. These growth regulatory peptides include the BMPs (Harris S., et al. (1994), supra). In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris S., et al. (1994), supra). Like alkaline phosphatase, osteocalcin and osteopontin, the BMPs are expressed by cultured osteoblasts as they proliferate and differentiate.
These data are, however, contrary to reports that dexamethasone and other inducers, such as BMPs, induce osteoblastic differentiation and stimulate osteocalcin mRNA (Bellows, C. G., et al., Develop Biol (1990) 140:132-38; Rickard, D. J., et al., Develop Biol (1994) 161:218-28). In addition, Ducy, P., et al., Nature (1996) 382:448-52 have recently reported that osteocalcin deficient mice exhibit a phenotype marked by increased bone formation and bones of improved functional quality, without impairment of bone resorption. Ducy, et al., state that their data suggest that osteocalcin antagonists may be of therapeutic use in conjunction with estrogen replacement therapy (for prevention or treatment of osteoporosis). However, there continues to be a need for additional treatments to stimulate bone growth or to mitigate bone loss.
Bisphosphonates, formerly called diphosphonates are compounds characterized by two C—P bonds. If the two bonds are located on the same carbon atom, resulting in a P—C—P structure, the compounds are called germinal bisphosphonates. They are therefore analogues of pyrophosphate that contain a carbon instead of an oxygen. There are a number of known pharmacologically active bisphosphonates including alendronate, Clodronate, etidronate, ibandronate, icadronate, pamidronate, risedronate, tiludronate and zoledronate. The main effect of these pharmacologically active bisphosphonates is to inhibit resorption both in vitro and in vivo. These effects are related to the marked affinity of these compounds for solid-phase calcium phosphate, on the surface of bone to which they bone strongly. In essence they target bone and elicit there pharmacological inhibition of osteoclast activity there. The mode of action of the bisphosphonates is still not completely elucidated. There is no doubt that their action in vivo is mediated mostly, if not completely, through mechanisms other than the physiochemical inhibition of crystal dissolution. There is a general consensus that the bisphosphonates act by inhibiting the activity of osteoclasts. Osteoclasts are inhibited when they come into contact with bisphosphonates-containing bone. This supports the hypothesis that bisphosphonates are deposited onto bone because of their strong affinity for the mineral, and that the osteoclasts are then inhibited when they start to engulf bisphosphonates-containing bone. The biochemical mechanisms by which bisphosphonates inhibit osteoclast activity are still unclear and may well be that more than one mechanism is operating.
The bisphosphonates investigated up to now appear to be absorbed, stored, and excreted unaltered in the body. Thus, bisphosphonates seem to be non-biodegradable, both in animals and in solution. Most of the pharmacokinetics data on the bisphosphonates have been obtained with etidronate, clodronate and pamidronate. The intestinal absorption lies between 1% and 10%. Between 20% and 50% of the absorbed bisphosphonate is localized to the bone, the remainder being rapidly excreted in the urine.
Although the nitrogen-containing bisphosphonates such as alendronate and pamidronate, have been shown to be effective in preventing the bone loss these drugs also appear capable of causing injury to the upper gastrointestinal tract and their have been several case reports of severe oesophagitis in patients treated with alendronate. Alendronate has also been shown to cause erosions and ulcers in the human stomach and to interfere with the healing of pre-existing lesions when given to healthy volunteers at doses that are prescribed for the treatment of osteoporosis and Pagets disease of bone.
The half-life of circulating bisphosphonates is short, in the rat only of the order of minutes. In man it is somewhat longer, about 2 hours. Acute, subacute, and chronic administration in several animal species have revealed little toxicity. Teratogenic, mitogenic and carcinogenic tests have been negative.
Other agents appear to operate by preventing the resorption of bone. Thus, U.S. Pat. No. 5,280,040 discloses compounds described as useful in the treatment of osteoporosis. These compounds putatively achieve this result by preventing bone resorption.
Nitric oxide (NO) has recently been shown to have profound effects on the metabolic activity of bone. This biologically active molecule generated biologically by a set of enzymes called nitric oxide synthases. Three known forms of the enzyme exist. Firstly, iNOS which is the inducible form where the expression of the enzyme and therefore the production of nitric oxide can be induced by a number of inflammatory stimuli. Secondly eNOS which is the constitutive form which is cannot be induced.
Endothelial dysfunction defined as the impaired ability of vascular endothelium to stimulate vasodilation plays a key role in the development of atherosclerosis and in various pathological conditions which predispose to atherosclerosis, such as hypercholesterolemia, hypertension, type 2 diabetes, hyperhomocyst (e) inemia and chronic renal failure. The major cause of the endothelial dysfunction is decreased bioavailability of nitric oxide (NO), a potent biological vasodilator produced in vascular endothelium from L-arginine by the endothelial NO synthase (eNOS). In vascular diseases, the bioavailability of NO can be impaired by various mechanisms, including decreased NO production by eNOS, and/or enhanced NO breakdown due to increased oxidative stress. The deactivation of eNOS is often associated with elevated plasma levels of its endogenous inhibitor, N(G) N(G)-dimethyl-L-arginine (ADMA). In hypercholesterolemia, a systemic deficit of NO may also increase the levels of low density lipoproteins (LDL) by modulating its synthesis and metabolism by the liver, as suggested by recent in vivo and in vitro studies using organic NO donors. Therapeutic strategies aiming to reduce the risk of vascular diseases by increasing bioavailability of NO continue to be developed.
Nitric oxide (NO) is a free radical which has important effects on bone cell function. The endothelial isoform of nitric oxide synthase (eNOS) is widely expressed in bone on a constitutive basis, whereas inducible NOS is only expressed in response to inflammatory stimuli. It is currently unclear whether neuronal NOS is expressed by bone cells. Pro-inflammatory cytokines such as IL-1 and TNF cause activation of the iNOS pathway in bone cells and NO derived from this pathway potentiates cytokine and inflammation induced bone loss. These actions of NO are relevant to the pathogenesis of osteoporosis in inflammatory diseases such as rheumatoid arthritis, which are characterized by increased NO production and cytokine activation. Interferon gamma is a particularly potent stimulator of NO production when combined with other cytokines, causing very high concentrations of NO to be produced. These high levels of NO inhibit bone resorption and formation and may act to suppress bone turnover in severe inflammation. The eNOS isoform seems to play a key role in regulating osteoblast activity and bone formation since eNOS knockout mice have osteoporosis due to defective bone formation. Other studies have indicated that the NO derived from the eNOS pathway acts as a mediator of the effects of estrogen in bone. eNOS also mediates the effects of mechanical loading on the skeleton where it acts along with prostaglandins, to promote bone formation and suppress bone resorption. Pharmacological NO donors have been shown to increase bone mass in experimental animals and preliminary evidence suggests that these agents may also influence bone turnover in man. These data indicate that the L-arginine/NO pathway represents a novel target for therapeutic intervention in the prevention and treatment of bone diseases.
Because of the importance of nitric oxide in many biological events numerous NO releasing compounds are now being synthesized. Many of these involve the use of linking NO to non-steroidal anti-inflammatory drugs (NSAIDS) such as flurbiprofen, ketoprofen, diclofenac and naproxen. These agent have been shown to spare the GI-tract from the undesired effects the NSAIDs by increasing blood flow and mucus secretion as well as reducing free radical generation in the stomach.